Assessing evolutionary patterns in uncultivable ciliates and their microbiomes by combining single-cell transcriptomics and single-cell genomics PROJECT SUMMARY Ciliates, the focus of the proposed work, have long been models for studies of genomes, resulting in the discovery of features including self-splicing RNA and telomeres. Like humans, ciliates have distinct germline and somatic genomes within every cell (i.e. individual) and the somatic ?macronuclear? genomes develop from zygotic nuclei through a complex series of epigenetically-regulated processes. Yet the bulk of studies on ciliates have focused on only a few cultivable (i.e. model) lineages such as Paramecium, Tetrahymena, and Oxytricha. Here, emerging single-cell transcriptomic and genomic techniques are combined with existing bioinformatics methods, including a taxon-rich phylogenomic pipeline, to address questions on the evolution of both the genomes and microbiomes of three uncultivable ciliate species ? Chilodonella uncinata, Halteria grandinella and Loxodes striatus ? that are estimated to have diverged approximately one billion years ago. By sampling natural populations of three target morphospecies, the proposed work will test two hypotheses: H1: Cryptic species of ciliates share similar morphology and protein-coding genes, but differ in germline/soma architecture; and H2: Ciliates contain species-specific microbiomes that contribute to the function of these organisms in their environments and that include human pathogens. Data collection will combine light microscopy and single-cell ?omics (transcriptomics and genomics). Data analyses will rely on existing bioinformatics tools, some of which have been developed in the PI?s lab. Interpretation of the resulting data will yield insights into: 1) genome evolution in ciliates, and more broadly eukaryotes; and 2) the biodiversity and possible function of endo- and ectosymbiotic bacteria in natural ciliate populations. The proposed work will take place at Smith College, an all women?s predominantly undergraduate institution. Each year, ~5-10 undergraduates will participate directly in the research and many more will learn about the results in courses taught by the PI. To support both the aims of this research project and the success of undergraduate participants, the PI will work with a postdoctoral fellow supported by this project. Because of the PI?s commitment to diversity and inclusion, ~50% of participants will be students of color and/or first-generation college students. The PI also publishes about 1 manuscript each year with undergraduate coauthor(s) and regularly takes students to national and international meetings. 1!